High-throughput, high-sensitivity detection or identification of molecules and other nanoscale objects is an important concept not only for medical diagnosis but also for drug-discovery, security, forensic and other applications. The most prominent tools for biological or chemical target detection or identification in a highly parallel fashion are microarrays. In recent years, DNA microarrays have been used extensively in genomic research, where they enabled massively-parallel interrogation of genetic code. However they are only capable of detecting or identifying complementary single stranded DNA or RNA molecules. In cases where other molecules or nanoscale complexes, for example proteins, have to be detected, more sophisticated devices, for example protein-arrays, sometimes also known in the art as protein microarrays, are required.
Protein microarrays present a significantly more difficult challenge than nucleic acid arrays, for example because of the complex nature of the proteome. Prospective protein arrays face several difficulties, in particular the identification of specific, high-affinity robust probe molecules that can bind to native proteins; the development of label-free sensing strategies for the detection of low abundance proteins in complex biological solutions; and the use of micron- or sub-micron-sized array features to enable high array densities of probe molecules.
In the art, methods of producing protein microarrays typically use surface immobilized antibodies and optical sensing of interactions with fluorescently labelled proteins. However, antibodies tend to lose their specificity and/or affinity when attached to surfaces. Further, antibodies are most often selected for binding to denatured, prokaryotically-expressed proteins either in animals or in vitro from phage display libraries. In addition, they predominantly recognize epitopes comprising conformationally constrained amino acid side chains in linear sequences. These limitations severely hinder the usefulness in applications where detection of conformationally dynamic native protein in complex bio-molecular mixtures, for example from cell lysates, is required.
Alternative probe molecules, for example RNA-aptamers, have been employed in protein arrays and these also suffer from the drawback that they usually have been selected for binding to prokaryotically expressed proteins that may not be correctly folded, and will not be post-translationally modified. The fluorescent dyes that are used to label proteins for subsequent detection of probe-target interactions are typically hydrophobic, and are likely to lead to conformational changes in labelled protein that may mask or destroy biologically relevant conformations.
A number of label-free detection strategies have been discussed in the prior art, including surface-plasmon resonance, mass spectroscopy, and atomic force microscopy-based techniques. Fabricating high-density arrays based on these strategies is however problematic and the instrumentation costs are significant.
US Patent Application No. 2005/023155 describes an apparatus and methods for the electrical detection of molecular interactions between a probe molecule and a protein or peptide target molecule, but without requiring the use of electrochemical or other reporters to obtain measurable signals. The invention described is a label-free detection system based on an electrochemical cell and conventional electrochemical impedance spectroscopy (EIS). The system is based on a glass capillary which closely resembles a conventional electrochemical cell. A problem with this approach is that array fabrication such as integrated array fabrication is extremely difficult.
International Patent Application WO2004/033724 describes a method of forming coatings of at least two different coating molecules on at least two electrodes. The method permits the preparation of nanoscale electrodes. The electrodes described therein are specifically coated using oligonucleotides and are designed for DNA and/or RNA detection. No protein or peptide based applications are demonstrated.
Prior art spotting techniques are typically available down to approximately 100 micrometers diameter of the spots of samples created. In the prior art, the smallest electrode which has previously been used has been approximately 150 micrometers in diameter. Each of these minimum size limitations represents a limitation imposed by the technical shortcomings of the state of the art.
The present invention seeks to overcome problem(s) associated with the prior art.